A typical angiosperm seed consists of three major components, the embryo, the endosperm and the maternal seed coat. Seed development begins with a double fertilization event, in which one sperm cell nucleus fuses with the egg cell nucleus to form the embryo, and a second sperm cell nucleus fuses with two central cell nuclei to form the endosperm. Embryo development itself can be separated into three developmental phases. The first phase of embryo development is one of cell division and morphogenesis, which serves to establish the major tissue types and organ systems of the mature plant. The second phase encompasses a period of rapid cell expansion and is characterized by the synthesis of storage reserves that sustain the embryo during germination and early seedling development. In the final phase of embryo development, the embryo becomes desiccated and enters into a period of developmental arrest or dormancy. All of the above events normally take place while the seed remains attached to the maternal plant.
Many plant species are capable of producing embryos in the absence of fertilization. This process of asexual embryo development may occur naturally, for example on the leaf margins of Bryophyllum (Yarborough, 1923) and Malaxis (Taylor, 1967), or within the ovule of apomictic plants (Koltunow, 1995). Apomixis refers to the production of a seed from the maternal ovule tissues in the absence of egg cell fertilization. Asexual embryo development may also be induced in vitro from gametophytic or somatic tissue (Mordhorst et al., 1997) or, as shown recently, may be induced by genetic modification of gene expression (Ogas et al., 1997; Lotan et al., 1998).
Three major mechanisms of apomixis, diplospory, apospory and adventitious embryony, have been observed. Each mechanism differs with respect to the source of the cell that gives rise to the embryo and with respect to the time during ovule development at which the apomictic process is initiated. Diplospory and apospory are considered gametophytic forms of apomixis as they involve the formation of diploid embryo sacs. Adventitious embryony does not involve the production of a mitotically-derived embryo sac.
In diplospory, the megaspore mother cell does not undergo normal meiosis, but rather divides mitotically to produce a diploid embryo sac instead of the normal haploid embryo sac. One of the cells of the embryo sac functions as the egg cell and divides parthenogenetically (without fertilization) to form an embryo. In some species the unreduced polar nuclei of the embryo sac may fuse to form the endosperm (autonomous endosperm production), the nutritive tissue of the seed, while in other species pollination is necessary for endosperm production (pseudogamy).
In aposporous apomicts, parthenogenic embryos are produced from additional cells, the aposporous initials, that differentiate from the nucellus. As with the megagametophyte of diplosporous species, the aposporous initial undergoes mitotic divisions to produce a diploid embryo sac. Aposporous embryos are not derived from the megagametophyte and can therefore co-exist within a single ovule with sexually-derived embryos. Autonomous production of endosperm is rare in aposporous species. Aposporous apomicts therefore depend on fertilization of the polar nuclei of a meiotically-derived embryo sac for the production of endosperm.
With adventitious embryony, embryos are formed directly from sporophytic ovule tissue, such as the integuments or nucellus, via parthenogenesis. Seeds derived from species exhibiting adventitious embryony generally contain multiple asexually-derived embryos and may also contain a single sexually-derived embryo. Plants exhibiting adventitious embryo also rely on the presence of a meiotically-derived embryo sac within the same ovule for endosperm formation.
In most plant species, the apomictic trait appears to be under the control of a single dominant locus. This locus may encode one or more developmental regulators, such as transcription factors, that in sexually reproducing plants function to initiate gene expression cascades leading to embryo sac and/or embryogenesis, but which are heterochronically or ectopically expressed in apomictic plants (Peacock, 1995; Koltunow, 1993; Koltunow et al, 1995).
Apomixis is a valuable trait for crop improvement since apomictic seeds give rise to clonal offspring and can therefore be used to genetically fix hybrid lines. The production of hybrid seed is a labour intensive and costly procedure as it involves maintaining populations of genetically pure parental lines, the use of separate pollen donor and male-sterile lines, and line isolation. Production of seed through apomixis avoids these problems in that once a hybrid has been produced, it can be maintained clonally, thereby eliminating the need to maintain and cross separate parental lines. The use of apomictic seed also provides a more cost effective method of multiplying vegetatively-propagated crops, as it eliminates the use of cuttings or tissue culture techniques to propagate lines, reduces the spread of diseases which are easily transmitted through vegetatively-propagated tissues, and in many species reduces the size of the propagule leading to lower shipping and planting costs.
Although apomixis occurs in a wide range of plant species, few crop species are apomictic. Attempts to introduce apomictic traits into crop species by introgression from wild relatives (Ozias-Akins, et al., 1993; WO 97/10704; WO 97/11167) or through crosses between related, but developmentally divergent sexual species (WO 98/33374), have not yielded marketable products. Other approaches have focused on the identification of gene sequences that may be used to identify or manipulate apomictic processes (WO 97/43427; WO 98/36090), however these approaches have not led to methods for the routine production of apomictic plants.
Mutagenesis approaches have also been attempted to convert sexually reproducing plants such as Arabidopsis thaliana (arabidopsis) into apomictic plants (Peacock et al., 1995). For example, a number of recessive “fertilization-independent seed” (fis) mutants have been identified that initiate partial embryo and/or endosperm at a low frequency in the absence of fertilization (Chaudhury et al., 1997). However, a number of additional parameters need to be modified in order to obtain true diploid apomictic seed using fis mutants.
Asexually-derived embryos can be induced to form in culture from many gametophytic and somatic plant tissues (Yeung, 1995). Somatic embryos can be obtained from culture of somatic tissues by treating them with plant growth regulators, such as auxins, or auxins in combination with cytokinins. Embryos can also be induced to form in culture from the gametophytic tissues of the ovule (gynogenesis) and the anther (androgenesis, pollen or microspore embryogenesis), either by the addition of plant growth regulators or by a simple stress treatment.
Several mutants have been identified that may be used to induce efficient production of embryos in vitro. These include recessive arabidopsis mutants with altered shoot meristems, for example primordia timing (pt), clavata (clv)1 and clv3, which were shown to enhance embryogenic callus formation when seedlings were germinated in the presence of auxin (Mordhorst et al., 1998). The altered expression of two arabidopsis genes, LEAFY COTYLEDON (LEC1; WO 98/37184, Lotan et al., 1998) and pickle, have been shown to promote the production of somatic embryos in the absence of added growth regulators. The LEC1 gene encodes a homologue of the HAP3 subunit of a CCAAT box-binding transcription factor (CBF). The LEC1 gene controls many aspects of zygotic embryo development including desiccation tolerance and cotyledon identity. Ectopic over-expression of the LEC1 gene in a led mutant background results in the production of 2 transgenic lines that occasionally form embryo-like structures on leaves. These embryo-like structures express genes, such as those encoding seed storage proteins and oil body proteins, which are normally preferentially expressed in developing embryos. Plants containing a recessive mutant PICKLE gene produce a thickened, primary root meristem. Mutant pickle roots produce embryo-forming callus when the root tissue is separated from the rest of the plant and placed on minimal medium without growth regulators (Ogas et al., 1997). Mutant pickle roots show morphological characteristics of developing seeds, such as oil bodies and, as with LEC1 over-expressers, accumulate genes preferentially expressed in developing seeds.
Efficient production of apomictic seed is only likely to be realised through the identification and subsequent modification of developmental regulators, such as transcription factors, that are known to activate gene expression cascades leading to embryogenesis in both sexually-reproducing and apomictic plants. The present invention addresses this need by providing methods for the production of apomictic seeds comprising ectopic over-expression of an embryo-expressed AP2 domain containing transcription factor, BNM3 or its homologs.
It is an object of the invention to overcome disadvantages of the prior art.
The above object is met by the combinations of features of the main claims, the sub-claims disclose further advantageous embodiments of the invention.